Various forms of influenza virus vaccine are currently available (e.g. see chapters 17 & 18 of reference 1). Vaccines are generally based either on live virus or on inactivated virus. Inactivated vaccines may be based on whole virions, ‘split’ virions, or on purified surface antigens.
Current influenza vaccines do not include an adjuvant, except for the FLUAD™ product from Novartis Vaccines, which includes an oil-in-water emulsion. Adjuvants have been used with experimental vaccines, however, with aluminium salts being used on several occasions, particularly for vaccines against pandemic strains. References 2 to 6 use an aluminum hydroxide adjuvant. Reference 7 used a mixture of aluminum hydroxide and aluminum phosphate. Reference 8 also described the use of aluminum salt adjuvants.
The usual assay for standardizing antigen content of influenza vaccines is the single radial immunodiffusion (“SRID”) assay [9,10], which was recommended by the WHO in 1978 to replace tests based on agglutination of erythrocytes. The assay is based on diffusion of influenza antigens into agarose gel containing specific anti-hemagglutinin (anti-HA) serum. As antigen diffuses outwards it meets a cognate antibody in the gel and initially forms a visible precipitate as a halo around the well. As diffusion continues, the concentration shifts towards excess substance and the precipitate dissolves. Diffusion continues further until the substance's concentration drops to allow precipitation again. Further diffusion allows precipitation, re-dissolving and re-precipitation until the substance is too low to re-dissolve the rim of the precipitate. The halo then stops increasing in diameter. The diameter of the final halo is directly proportional to the amount of HA antigen in the preparation. By comparing halos produced by an unknown preparation to those of a reference with known HA content, an antigen amount can be assigned to the unknown.
The adjuvanted vaccine in reference 5 was prepared by extemporaneously mixing antigen and adjuvant immediately prior to use. Antigen content had thus been standardized before adjuvant was introduced. If antigen and adjuvant are mixed at the stage of bulk vaccine manufacture, however, the SRID assay will have to be performed on adsorbed antigen. The inventors have found that influenza HA binds to aluminium salt adjuvants so tightly that it does not diffuse well into the SRID gel and so cannot easily be assayed directly by the standard procedure. Thus there is a need to provide improvements to the SRID assay which allow it to be used with pre-adsorbed vaccines.